Acylated compounds for the treatment of ocular pathologies

ABSTRACT

A therapeutic use of acylated piceid derivative compounds in ocular pathologies, in particular retinitis pigmentosa and in age-related macular degeneration, inter alia. A method of treating and/or preventing ocular pathologies, wherein the method includes administering to a patient in need of treatment a therapeutically effective amount of a compound of general formula (I) or any of its isomers, their pharmaceutically acceptable salts, esters, tautomers, polymorphs, or hydrates.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the therapeutic use of acylated piceidderivatives in ocular pathologies, particularly in retinal degeneration.The present invention can therefore be encompassed within the field ofpharmaceuticals.

Description of the Related Art

Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) area cause of blindness in the adult population due to the degeneration ofphotoreceptors and of the retinal pigment epithelium (RPE). RP is a raredisease, but the prevalence of AMD is quite high, estimated at 8-16% ofthe population between 50 and 64 years of age. Like for RP, no treatmentexists for the dry variety of AMD despite its high prevalence.

Patent application WO 2007020673 A1 discloses compounds derived fromglycosylated hydroxystilbenes that might be useful for the treatment ofsome ocular diseases. Acylated piceid (3-glucosyl resveratrol)derivatives have been proposed for the treatment of inflammatory boweldiseases (ES 2362065 A1) and as antibiotics (Selma M V et al., J. Agric.Food. Chem. 2012, 60(30), 7367-74).

Nevertheless, there are currently no effective treatments that canprevent or slow down the degenerative process of photoreceptors or RPAin dry AMD or RP. Therefore, it would be beneficial to search for newmolecules that are useful for the treatment or prevention of these andother ocular diseases involving retinal degeneration.

SUMMARY OF THE INVENTION

The present invention provides compounds that are useful for themanufacture of a pharmaceutical composition for the treatment orprevention of ocular diseases. To this end, it is demonstrated in theexamples how some acylated derivatives of resveratrol induce theprotection of the photoreceptors in two animal models of retinaldegeneration.

In a first aspect, the present invention relates to the use of compoundsof the general formula (I)

or any of its isomers, or any of its salts, preferably anypharmaceutically acceptable salt, esters, tautomers, polymorphs,pharmaceutically acceptable hydrates, or prodrugs, derivatives,solvates, or the like, or any combination thereof,

where:

R₁ is a C₁-C₂₂ alkyl group or a C₂-C₂₂ alkenyl group, with R₁ preferablybeing a C₁-C₂₂ alkyl group;

for the preparation of a pharmaceutical composition for the preventionand/or treatment of ocular pathologies.

In a preferred embodiment, R₁ is a C₂-C₂₀ alkyl group, more preferably aC₃-C₁₇ alkyl group.

In the present invention, the term “alkyl” refers to aliphatic, linear,or branched chains having from 1 to 22 carbon atoms, e.g., methyl,ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl,n-hexyl, etc. The alkyl group preferably has between 2 and 20 carbonatoms, and more preferably between 3 and 17 carbon atoms.

In the present invention, the term “alkenyl” refers to unsaturated,linear, or branched aliphatic chains having from 2 to 22 carbon atomsand having between one and six unsaturations depending on the number ofcarbons, for example, without limitation to vinyl, allyl, oleyl,linoleyl, linolenyl, eicosapentaenoyl, docosahexaenoyl, etc.

In a preferred embodiment, the compounds of the invention can beselected from among:

trans-resveratrol-3-O-(6′-O-butanoyl)-β-D-glucopyranoside (1),

trans-resveratrol-3-O-(6′-O-octanoyl)-β-D-glucopyranoside (2),

trans-resveratrol-3-O-(6′-O-hexadecanoyl)-β-D-glucopyranoside (3),

trans-resveratrol-3-O-(6′-O-octadecanoyl)-β-D-glucopyranoside (4), andany combination thereof.

The pharmaceutical composition of the present invention can beformulated for administration in a variety of manners that are known inthe art. Examples of preparations include any solid composition(tablets, pills, capsules, granules, etc.) or liquid composition(solutions, suspensions, or emulsions) for oral, topical, or parenteraladministration. The composition of the present invention can also be inthe form of sustained-release drug formulations or any otherconventional delivery system, and can thus be contained, withoutlimitation thereto, in nanoparticles, liposomes, or nanospheres, in apolymeric material, in a biodegradable or non-biodegradable implant, orin biodegradable microparticles such as biodegradable microspheres, forexample.

Such a composition and/or its formulations can be administered to ananimal, including a mammal and hence to a human, in a variety ofmanners, including but not limited to intraperitoneal, intravenous,intradermal, intraspinal, intrastromal, intraarticular, intrasynovial,intrathecal, intralesional, intraarterial, intramuscular, intranasal,intracranial, subcutaneous, intraorbital (sub-retinal, intravitreal),intracapsular, topical, or percutaneous administration, or by means oftransdermal patches, nasal spray, surgical implant, internal surgicalpainting, or infusion pump.

In an embodiment that is even more preferred, the composition of theinvention is formulated for ophthalmic administration. The expression“formulated for ophthalmic administration” refers to a formulation thatenables the composition of the invention to be administeredocularly—including, but without limitation thereto, topically orintraocularly—without such administration adversely affecting theproperties, e.g., optical and/or physiological properties, of the eye.

Examples of the composition of the invention that are formulated forophthalmic administration include but are not limited to saidcomposition in combination with water, salts, a polymeric liquid orsemi-solid vehicle, a phosphate buffer, or any other ophthalmicallyacceptable liquid vehicle thereof that is known in the art.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a system for the controlled release of the compoundsof the invention; more preferably, they comprise cyclodextrins and evenmore preferably 2-hydroxypropyl beta-cyclodextrin.

Therefore, another aspect of the present invention relates to anophthalmological composition comprising at least one compound of theformula (I) as described above together with a controlled-releasesystem, particularly a cyclodextrin, and more specifically2-hydroxypropyl beta-cyclodextrin.

In another aspect, the present invention relates to a method fortreating and/or preventing ocular pathology in a mammal, preferably ahuman, comprising the administration of a therapeutically effectiveamount of a composition of the general formula (I) as described above.

The dosage for obtaining a therapeutically effective amount depends on avariety of factors, such as the age, weight, sex, or tolerance of theindividual to whom the composition of the invention is to beadministered, for example. In the meaning used in this description, theterm “therapeutically effective amount” refers to the amount of thecomposition (I) calculated to produce the desired effect and, ingeneral, will be determined inter alia by the characteristics of thecomposition, the age, condition, and history of the patient, theseverity of the alteration or disorder, and the route and frequency ofadministration.

The ocular pathologies of the present invention can be selected fromamong:

1. Hereditary degenerative pathologies of the retina, includingretinitis pigmentosa and all those pathologies within the group ofretinal dystrophies which include, but are not limited to: autosomaldominant retinitis pigmentosa, autosomal recessive retinitis pigmentosa,retinitis pigmentosa linked to the X chromosome, sporadic retinitispigmentosa, retinitis pigmentosa associated with other syndromes,Cokayne syndrome, cone dystrophies, cone and rod degeneration, Leber'scongenital amaurosis, retinitis punctata albescens, choroideremia,choroidal and retinal gyrate atrophy, choroidal generalized dystrophy,juvenile retinoschisis, Wagner's vitreoretinal degeneration, autosomaldominant vitreoretinal choroidopathy, fundus albipunctatus, Stargardt'sdisease, Best vitelliform macular dystrophy, Usher syndrome,Bardet-Biedl syndrome, fenestrated macular dystrophy, Sorsbypseudoinflammatory macular dystrophy, dominant drusen, among otherretinal dystrophies involving degeneration of photoreceptors and/or EPR.

2. Early- and/or late-onset age-related (wet or neovascular) and/ornon-exudative (dry or atrophic) macular degeneration; as well as othermacular diseases such as: epiretinal membrane, macular hole, and macularedema, etc.

3. Degenerative inflammatory pathologies of the retina and the opticnerve of infectious, immune, vascular, traumatic, post-surgical, orteratogenic origin such as: ocular toxoplasmosis, cryptococcosis, lupus,Behcet's disease, diabetic retinopathy, ischemic optic neuropathy, opticneuritis, detachment of the retina, vitrectomy, internal limitingmembrane peeling, surgery, retinoblastoma, etc.

4. Ocular pathologies that increase intraocular pressure and thatinvolve degeneration of the ganglion cells of the retina and atrophy ofthe optic nerve such as: open-angle or closed-angle glaucoma.

5. Neurological pathologies that are accompanied by degenerative lesionsof the retinal or optic nerve such as: amyotrophic lateral sclerosis,multiple sclerosis, Parkinson's disease, Alzheimer's disease, Graefesyndrome, Hallgren syndrome, Hallen Vorden Spatz syndrome, autosomaldominant cerebellar ataxia, and pigmentary degeneration of the retina.

6. Other ocular pathologies that occur with inflammation of the cornea,choroid, ciliary body of any etiology such as: uveitis, scleritis,keratoconjunctivitis, corneal transplant surgery, cataract surgery.

7. Other ocular or neurodegenerative pathologies that involveinfiltration of microglia or shortening of telomeres due to cellularaging or oxidative stress.

In one preferred embodiment, the ocular pathologies are selected fromamong retinitis pigmentosa and age-related macular degeneration.

Throughout the description and the claims, the expression “comprises”and variants thereof are not intended to exclude other technicalfeatures, additives, components, or steps. For those skilled in the art,other objects, advantages, and features of the invention will beapparent in part from the description and in part from the practicing ofthe invention. The following examples and figures are provided for thesake of illustration and are not intended to limit the scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cell viability in neuroblastoma SH-SY5Y after damage with H₂O₂and treatment with the different compounds 1-4. The controls are—H₂O₂=1% DMSO solution; +H₂O₂═H₂O₂ in 1% DMSO; RES 10μM+H₂O₂=resveratrol 10 μM in H₂O₂ in 1% DMSO.

FIG. 2. Cell viability in RAW macrophages after inflammation producedwith LPS and treatment with the different compounds 1-2. The controlsare -LPS=1% DMSO solution; +LPS=1% DMSO solution with LPS; RES 10μM+LPS=resveratrol 10 μM in 1% DMSO solution with LPS.

FIG. 3. Concentration of TNF-alpha in culture medium after inflammationby LPS in RAW macrophages and treatment with compound 2 and RES control.The controls are LPS alone=1% DMSO solution with LPS; DMSO only=1% DMSOsolution.

FIG. 4. Concentration of NO in culture medium after inflammation by LPSin RAW macrophages and treatment with compound 2 and RES control. Thecontrols are LPS alone=1% DMSO solution with LPS; DMSO only=1% DMSOsolution.

FIG. 5. Concentration of IL6 in culture medium after inflammation by LPSin RAW macrophages and treatment with compound 2 and RES control. Thecontrols are LPS alone=1% DMSO solution with LPS; DMSO only=1% DMSOsolution.

FIG. 6. AChE activity with respect to the control of compound 2 and theRES control.

FIG. 7. Fundus of normal c57/bl6 mouse (WT) (A), untreated rd10 mouse(B), and injected with 5 mM of compound 2 (C) at 15 days aftersubretinal administration of the compounds. The absence of pigment inthe fundus of the mouse treated with compound 2 is observed.

FIG. 8. Electroretinogram of normal c57/bl6 mouse (WT), untreated rd10mouse, DMSO vehicle 5%, 5 mM resveratrol (RES) and 5 mM compound 2 at 15days after subretinal injection. The amplitude of the a- and b-waves waspreserved in the group treated with compound 2 in a greater proportionthan in the rd10 mice that were untreated or treated with RES or with 5%DMSO vehicle.

FIG. 9. Amplitude of the b-wave in untreated rd10 mice, rd10 micetreated with 5% DMSO vehicle, with 5 mM RES, and with 5 mM compound 2 at15 days after administration of the subretinal injections. Each barrepresents the average of the b-wave in 6 mice per group±the standarderror of the mean. The amplitude of the b-wave was significantlyincreased in the mice treated with compound 2 in the dark-adapted ERGwith flash intensities of 0.2, 1, 3 and 10 cd xs/m². The statisticalanalysis was performed by one-way ANOVA with post hoc DMS test *p<0.05;#p<0.01.

FIG. 10. Immunofluorescence of normal c57/bl6 mouse (WT) (A, G, M),untreated rd10 mouse (B, H, N), or rd10 mice treated with vehicle (C, I,O), with 5 mM RES (D, J, P) and with 5 mM compound 2 (F, L, R) at 15days after administration of the subretinal injections. The sections ofthe retina were immunostained with anti-rhodopsin antibodies (red) tolabel the rods and anti-opsin (green) to label the cones. The nucleiwere stained with DAPI. In all of the groups that were treated withSIRT1 activators, more labeling of rhodopsin and opsin was observed thanin the untreated group, but the signal of this immunolabeling was moreintense in the group treated with compound 2. Likewise, the thickness ofthe outer nuclear layer (ONL) was greater than in the group treated withRES.

FIG. 11. Fundus and autofluorescence of the retina in normal mice (WTcontrol), untreated Prpf31^(A216P/+) mice (KI control), andPrpf31^(A216P/+) mice treated with eye drops administered daily for twoand a half months containing 2 mM resveratrol (KI RES) or 2 mM compound2 diluted in a 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KI 2)or only with the 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KICYCLE). Both WT and KI mice that did not receive any treatment (WTcontrol and KI control) were used as controls. All the groups weresubjected to 5,000 lux for 3 h and the evaluations were made 15 daysafter inducing light damage, the eye drops having been continued duringthis 2-week period. In the KI control, KI CYCLE, and KI RES groups,areas with autofluorescent punctiform yellow-white lesions (arrows) wereobserved which can be seen clearly in the enlargement of the image inthe lower row. Moreover, areas of focal atrophy (arrowheads) wereobserved in these mice. Fundi with this type of characteristic wereconsidered abnormal. In the majority of the fundi of the WT control andKI 2 groups, this type of lesion was not observed, and these fundi wereclassified as normal.

FIG. 12. Thickness of the retina quantified by means of OCT in normalmice (WT control), untreated Prpf31^(A216P1/+) mice (KI control), andPrpf31^(A216P/+) mice treated with eye drops administered daily for twoand a half months containing 2 mM resveratrol (KI RES) or 2 mM compound2 diluted in a 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KI 2)or only with the 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KICYCLE). Both WT and KI mice that did not receive any treatment (WTcontrol and KI control) were used as controls. All the groups weresubjected to 5,000 lux for 3 h and the evaluations were made 15 daysafter inducing light damage, the eye drops having been continued duringthis 2-week period. The retinal maps (A) show the thickness of theretina in 9 concentric areas at 1, 2.22, and 3.45 mm and represented bya colorimetric scale (B). The data were quantified and are shown in (C).The bars represent averages of retinal thickness measured in μm+/−thestandard error (WT control n=5, rest of the groups n=10). The normalityof the data was confirmed by the Kolmogorov-Smirnov test (P=0.98); todetermine differences in the means of the groups, one-way ANOVA(P<0.001) and post hoc LSD tests were performed. *P<0.001 KI control vs.KI RES or KI control vs KI 2.

FIG. 13. Evaluation of the amplitude of the c-wave in normal mice (WTcontrol), untreated Prpf31^(A216P/+) mice (KI control), andPrpf31^(A216P/+) mice treated with eye drops administered daily for twoand a half months containing 2 mM resveratrol (KI RES) or 2 mM compound2 diluted in a 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KI 2)or only with the 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KICYCLE). Both WT and KI mice that did not receive any treatment (WTcontrol and KI control) were used as controls. All the groups weresubjected to 5,000 lux for 3 h and the evaluations were made 15 daysafter inducing light damage, the eye drops having been continued duringthis 2-week period. The amplitude of the c-wave was measured by ERG (A)and then the amplitude of the b-wave was quantified (A), and thec-/b-wave ratio (C) was also estimated. The bars represent averages ofthe c-/b-wave ratio +/−the standard error (WT control n=5, rest of thegroups n=10). The normality of the data was confirmed by theKolmogorov-Smirnov test (P=0.43); to determine differences in the meansof the groups, one-way ANOVA (P<0.05) and post hoc LSD tests wereperformed. *P<0.01 KI control vs KI 2.

FIG. 14. Optomotor test showing the visual acuity curve in normal mice(WT control), untreated Prpf31^(A216P/+) mice (KI control), andPrpf31^(A216P/+) mice treated with eye drops administered daily for twoand a half months containing 2 mM resveratrol (KI RES) or 2 mM compound2 diluted in a 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KI 2)or only with the 13% hydroxypropyl-beta-cyclodextrin vehicle in PBS (KICYCLE). Both WT and KI mice that did not receive any treatment (WTcontrol and KI control) were used as controls. All the groups weresubjected to 5,000 lux for 3 h and the evaluations were made 15 daysafter inducing light damage, the eye drops having been continued duringthis 2-week period. The optomotor test data were quantified and areshown in the chart. Each point represents averages of the number ofpositive responses +/−the standard error (WT control n=5, rest of thegroups n=10). The normality of the data was confirmed by theKolmogorov-Smirnov test in all spatial frequencies except for 0.031 and0.061. To determine differences in the means of the groups, two-wayANOVA was performed, correlating both the different frequencies andgroups (P<0.001) and the post hoc LSD test. *P<0.05 KI control vs. KIRES or **<0.001 KI control vs KI 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Examples

The invention will be illustrated below by means of tests conducted bythe inventors that demonstrate the effectiveness of the product of theinvention.

Example 1. In Vitro Neuroprotection Assays

The SH-SY5Y neuroblastoma cell line was cultured in petri dishespre-treated with collagen (100 μg/ml) with F12 medium supplemented withpenicillin/streptomycin and 10% inactivated fetal bovine serum.

Cell viability assays with neurons were prepared in 96-well platespre-treated with collagen by seeding 20,000 cells/well in a volume of100 μl and incubating the cells for 24 h before the addition of thecompounds. The compounds to be tested were dissolved in DMSO and addedin three different concentrations (1, 10 and 100 μM) in order todetermine their toxicity. The final percentage of DMSO in each well wasadjusted to 1%. The cell viability was evaluated 24 h after the additionof the compounds by means of the MTT assay according to themanufacturer's method. Mean values and standard deviations werecalculated from at least 8 different measurements from severalindependent experiments.

Neuroprotection assays. The neurons were cultured and seeded in the samemanner as for the cell viability assay. The compounds to be tested weredissolved in DMSO and added to three different concentrations (1, 10 and100 μM) and, after 10-minute incubation, hydrogen peroxide (100 μM) wasadded to the medium. The final percentage of DMSO in each well wasadjusted to 1%. The cell viability was evaluated 24 h after the additionof the compounds by means of the MTT assay according to themanufacturer's method. Mean values and standard deviations werecalculated from at least 8 different measurements from severalindependent experiments. Neuronal recovery was calculated by normalizingthe results of the neuronal viability experiments after the addition ofour compounds and H₂O₂ to the positive control of each experiment(neurons+H₂O₂).

It is observed that the RES 10 μM control recovers up to 50% of cellviability (indicated by the arrow in FIG. 1). In contrast, acylatedpiceid derivatives (1, 2, 3 and 4) recover between 70 and 200% ofviability at concentrations between 1 and 10 μM. All of those at 100 μMappear to exhibit toxicity.

Example 2. In Vitro Inflammation Assays

RAW 264.7 macrophages were cultured in P75 with high-glucose DMEmsupplemented with penicillin/streptomycin and 10% inactivated fetalbovine serum.

The cell viability assays with RAW macrophages were prepared in 96-wellplates by seeding 25,000 cells/well in a volume of 100 μl and incubatingthe cells for 4 h before the addition of the compounds. The compounds tobe tested were dissolved in DMSO and added in three differentconcentrations (1, 10 and 100 μM) in order to determine their toxicity.The final percentage of DMSO in each well was adjusted to 1%. The cellviability was evaluated 24 h after the addition of the compounds bymeans of the MTT assay according to the manufacturer's method. Meanvalues and standard deviations were calculated from at least 8 differentmeasurements from several independent experiments.

Testing of mitigation of damage caused by the addition of LPS. The RAW264.7 macrophages were cultured according to the procedure describedabove. The compounds to be tested were dissolved in DMSO and added tothree different concentrations (1, 10 and 100 μM) and, after 10-minuteincubation, LPS (100 ng/ml) was added to the medium. The finalpercentage of DMSO in each well was adjusted to 1%. The cell viabilitywas evaluated 24 h after the addition of the compounds by means of theMTT assay according to the manufacturer's method. Mean values andstandard deviations were calculated from at least 8 differentmeasurements from several independent experiments.

It is observed that the RES 10 μM control recovers up to 82% of cellviability (indicated by the arrow in FIG. 2). Compound 2 recoversbetween 80 and 140% of viability at concentrations between 1 and 100 μM.

Example 3. Measurements of Inflammation Parameters (Cytokines) in Assaywith LPS

To determine the production of cytokines, 5×10⁵ RAW 264.7 macrophageswere seeded in 24-well plates (0.5 ml per well). The compounds to betested were then added (10 μM), and the macrophages were eitherstimulated or not through the addition of LPS (1 μg/ml) to the culturemedium. After 24 hours, the levels of IL-6 and TNF-α were measured inthe supernatants by ELISA using the capture and biotinylated antibodiesfrom BD PharMingen and PrepoTech (Gonzalez-Rey et al., Gut. 2009;58:929-939; Sanchez et al., Stem Cells. 2011; 29:251-262). The levels ofNO in the supernatants at 24 hours were measured indirectly bydetermining the nitrite concentration in the medium using the Griessreagent (Anderson et al., Gut. 2013; 62:1131-1141). A minimum of twoindependent experiments and three replicates per experiment wereperformed for each measured value. The values are expressed as themean±standard deviation.

In the previous assay, the levels of various inflammation parameterswere measured (TNF-α, NO, and IL-6) by ELISA after treatment with RES orwith compound 2 (FIGS. 3, 4, and 5).

It is observed that the control of RES 10 μM significantly decreasesinflammatory parameters (TNF-α, NO, and IL-6) (indicated by the bar inbold). Compound 2 decreases TNF-α and NOT even more than RES andsimilarly to IL-6.

Example 4. Evaluation of the Neuroprotective Capacity of VariousSilylated Compounds in a Model of Neurodegeneration in Zebrafish LarvaeInduced by Pentylenetetrazole (PTZ)

The objective of this assay was to analyze the protective effect ofcompound 2 and of the resveratrol (RES) control in a model ofneurotoxicity induced by the neurotoxin pentylenetetrazole (PTZ). As anexperimental model, the zebrafish larva (Danio rerio) was used to studythe effect of the compounds on acetylcholinesterase activity (AChE) inlarvae at 5 days post-fertilization (dpf).

Studies of the central nervous system (CNS) in zebrafish (Kimmel et al.,Dev Dyn, 1995 July; 203(3):253-310), show that, at 24 hours ofdevelopment, the brain of the embryo has already segmented and somestructures such as the neural tube, the notochord and the somites(muscle, and bone precursors). At 5 days post-fertilization (5 dpf), theanimal has formed sensory organs such as eyes and otoliths. In addition,the heart, liver, kidneys, and pancreas, as well as the circulatory,digestive, and nervous systems are fully functional. At this time, theanimal is able to respond to visual, olfactory, and mechanical stimuliand begins the search for food.

AChE is an enzyme that degrades through its hydrolytic activity theneurotransmitter acetylcholine in choline and an acetate group. It isfound chiefly in neuromuscular junctions and in the cholinergic nervoussystem, where its activity has the function of ending synaptictransmission (Behra et al., Nat Neurosci. 2002 February; 5(2): 111-8).Acetylcholine is a neurotransmitter involved in the control of movementand is an important modulator of cognitive functions such as learningand memory (Hasselmo et al., Neuropsychopharmacology. 2011 January;36(1): 52-73). Adequate levels of acetylcholinesterase thus reflect ahealthy neuronal state.

Zebrafish embryos were seeded in 50 ml of dilution water (AD) in a Petridish and grown to 5 dpf (larval stage). Only those larvae that did notexhibit any type of external anomaly were used to perform the assay.Next, the larvae were transferred using a Pasteur pipette to a 24-wellmicroplate, so that each well contained five larvae, making tenreplicates per condition. First, the pre-treatment of the 5 dpf larvaewas performed. For this, the larvae were incubated at 26±1° C. for 1hour in a volume of 2 ml of AD for the two control groups (Control andControl+PTZ), of physostigmine (Phys) 20 μM, which is a commercialinhibitor of the enzyme AChE for the Phys group, and of the testcompounds at a concentration of 10 μM. A medium exchange was thencarried out, and the larvae were incubated with the compounds incombination with 5 mM PTZ for 6 hours at 26±1° C. After this incubationperiod, all of the larvae were examined, and it was determined that thegeneral state of the larvae was totally normal, without any visibleanomaly or anomalous behavior. Finally, the larvae were processed forthe analysis of the AChE activity.

Determination of AChE levels. Once the experimental period wascompleted, larvae processing was carried out for the determination ofAChE according to the technical study protocol (Measurement ofacetylcholinesterase activity (AChE) in cell cultures and zebrafishlarvae). The larvae were homogenized mechanically, and the samples werecentrifuged to obtain the supernatant, which were used to determine thelevels of the AChE enzyme as a function of the treatments administered.In addition, the determination of total protein of each experimentalgroup was carried out according to the technical study protocol(Quantification of protein by BCA, as a normalization process). Finally,the AChE levels determined in the control group were taken as areference measurement and deemed to be 100%.

Compound 2 significantly prevents the decrease in AChE activity inducedby PTZ in the 5 dpf larvae, showing a clear neuroprotective effect thatis greater than that observed for RES (see FIG. 6).

Example 5.—Toxicity Assay in Zebrafish

Twenty zebrafish embryos were incubated for each concentration ofcompound to be tested (1 to 4). The concentrations used of each compoundare 0.1 μM, 1 μM, 10 μM, 100 μM and 1 mM. The incubation period goesfrom 0 to 96 hours post fertilization (hpf). A negative control of 1%DMSO and a positive control of N, N-diethylaminobenzaldehyde (DEAB) areincluded. After the incubation period, the LC50 (lethal dose for 50% ofthe animals) is calculated.

The zebrafish embryo toxicity assay was carried out for compound 2 andthe resveratrol (RES) control, and both exhibited acceptable toxicity(LC50<140 uM) and (LC50<1000 uM), respectively.

Example 6. Assays in Rd10 Mice, Retinal Degeneration Model

The rd10 mouse is a model of retinitis pigmentosa due to a mutation inthe Pde6b gene that results in the degeneration of the photoreceptorsthat begins on postnatal day 15-16 (P15-16) and is completed within onemonth (P30). In this model, the degeneration begins in the rods and thencontinues with cones and finally the retinal pigment epithelium (RPE).The degeneration in this model occurs as a consequence of the loss ofthe function of Pde6b, which ultimately leads to a continuous entry ofCa⁺⁺ in the cones with a significant production of reactive oxygenspecies (ROS), infiltration of macrophages, and activation ofpro-apoptotic proteins. At the histological level, a loss of nuclei isobserved in the outer nuclear layer of the retina (the layer in whichphotoreceptor nuclei are located), as well as the loss of the specificmarkers of rods (rhodopsin) and cones (opsin). The electroretinogram(ERG), which measures the electrical response of the retina to visualstimuli, is markedly affected in rd10 mice, with progressive loss of theamplitude of the a- and b-waves of the ERG. Finally, the fundus used toevaluate the macroscopic morphology of the bottom of the eye exhibitschanges at one month of age in the rd10 mice, with accumulation of darkpigments in the periphery due to the degeneration of the RPE. Toevaluate the protective effect of our compounds on the retina, we firstused the rd10 model. Rd10 homozygous mice were injected in thesubretinal space at P13 (two to three days before the onset ofdegeneration) with 1 μl of vehicle (5% DMSO in PBS) or resveratrol RES(5 mM), as well as compound 2 (5 mM). Then, 15 days after having beeninjected, fundus, ERG, and histological changes were evaluated in theuntreated mice, those treated with the vehicle, and those treated withRES and compound 2. Evaluation of the fundus revealed a lower amount ofpigment accumulation in the retinas of mice treated with compound 2compared to the untreated mice (FIG. 7). The ERG in the treated mice wasalso evaluated 15 days after the injections. The amplitude of the a- andb-waves in the ERGs were quantified, and the statistical analysis wasperformed using two-way ANOVA and post hoc DMS. A value of p<0.05 wasconsidered significant. The b-wave amplitude in the ERG of dark-adaptedmice (flash intensities of 0.2, 1, 3, and 10×cd s/m²) underwent asignificant increase in the group of mice treated with compound 2compared to the controls (FIGS. 8 and 9). Finally, theimmunofluorescence study was performed in order to observe the presenceof markers of rods (rhodopsin) and cones (opsin) (FIG. 10). The numberof photoreceptor nuclei present in the outer nuclear layer (ONL), aswell as the immunostaining of rhodopsin, was much greater in rd10 micetreated with compound 2 than in those treated with RES and in thecontrols.

Example 7.—Assays on Prpf31 Mice, RPE Retinal Degeneration Model)

Prpf31^(A216P/+) mice are a useful model for assessing the degenerationof the RPE. This model is characterized by an increase in the thicknessof the Bruch membrane and accumulation of esterified cholesterol betweensaid membrane and the RPE produced by a defect in the splicing of themRNA of relevant genes of the visual cycle, such as RPE65 and RDH12. Theaccumulation of toxic products derived from retinol leads to an increasein ROS, infiltration of macrophages, and primary degeneration of theRPE. The degeneration of the RPE occurs late, at approximately 8 monthsof age. The phenotypic characteristics of this model can be exacerbatedby exposure to intense light and are characterized primarily byaccumulation of pseudo-drusenoid lesions and regions of atrophy observedin the fundus, with a loss of visual acuity and perception of contrastbeing observed, as well as a decrease in the amplitude of the c-wave ofthe ERG and a decrease in the thickness of the retina.

To evaluate the protective effect of the administration of our compoundsin eye drops, we used heterozygous 6 month-old Prpf31^(A216P/+) (KI)mice that were treated topically with 20 μl of the studied compoundsapplied directly on the corneal surface daily (L-V) for two and a halfmonths. The compounds used were RES and compound 2, which were dilutedin a solution of 13% hydroxypropyl-beta-cyclodextrin (CYCLO) in order toobtain a final concentration of 2 mM of each compound. After two and ahalf months of application of the eye drops with CICLO, RES, or compound2, the mutant KI mice were exposed to a light source of 5,000 lux for 3hours. Normal (WT) and KI mice were also exposed to intense light astest control without receiving any treatment. Subsequently, the fundusof the animals was evaluated, in addition to the visual acuity by meansof the optomotor test, the thickness of the retina by means of opticalcoherence tomography (OCT), and the amplitude of the c-wave by ERG.

Evaluation of the Fundus:

Two categories were used to quantify the macroscopic characteristics ofthe fundus: abnormal and normal fundus. The fundi categorized asabnormal were those in which the presence of autofluorescentpseudo-drusenoid lesions was observed (FIG. 11; arrows) or with regionsof focal atrophy (FIG. 11; arrowheads). The percentage of abnormal fundiin the WT mice exposed to light (WT control) was 20%, and in theuntreated KI (KI control) it was 90%. Meanwhile, this percentage wasreduced to 30% in mice treated with compound 2 (KI 2) and only to 60% inboth mice treated with CYCLE (KI CYCLE) and RES (KI RES) (table 1). Whenperforming the statistical analysis for each dichotomous variable, weobserved that there is a statistically significant relationship betweenthe presence of normal fundi and mice treated with compound 2 whencompared with the control KI group (chi-square=7.5; P<0.01, Fisher'sexact test P<0.05) and with a probability of obtaining a normal fundusin the group treated with compound 2 of seven times greater than in thecontrol KI (normal RR=7, IC95%=1.044-46.949). These values were similarto those observed when comparing WT control and KI control(chi-square=7.35, P<0.01, Fisher's exact test P<0.05) and with aprobability of obtaining a normal fundus in the WT control group ofeight times greater than in the control KI (normal RR=8;IC95%=1.184-54.043). In both the group treated with CICLO and thattreated with RES, no statistically significant association was observedwhen comparing each one to the control KI group. The conclusion of thisexperiment is that treatment with compound 2 prevented the formation ofautofluorescent pseudo-drusenoid lesions and regions of focal atrophy inthe Prpf31^(A216P/+) mouse.

TABLE 1 Quantification of the number of abnormal and normal fundi in WTand Prpf31^(A216/+) (KI) mice without treatment or when treated with 13%CYCLE, RES, and compound 2. Fundus/Groups Abnormal (%) Normal (%) TotalWT control  1 (20)  4 (80)^(#) 5 KI control  9 (90)  1 (10) 10 KI CYCLE 6 (60)  4 (40) 10 KI RES  6 (60)  4 (40) 10 KI 2  3 (30)  7 (70)^(*) 10Total (%) 25 (55.6) 20 (44.4) 45 ^(*)KI 2 vs. KI control (chi-square =7.5; P < 0.01; Fisher′s exact test P < 0.05; RR normal = 7; IC95% =1.044-46.949). ^(#)WT control vs KI control (chi-square = 7.35, P <0.01, Fisher′s exact test P < 0.05, normal RR = 8, 95% CI =1.184-54.043).

Thickness of the Retina:

To evaluate the thickness of the retina in vivo, a Stratus OCT (Zeiss)was used with a scanner adjusted to 6 lines of 3.45 mm each arrangedradially. Six measurements were performed in each eye, upon which theretinal map was prepared (FIG. 12) with the thickness of the retinaquantified from the ganglion cell layer to the RPE. The thickness of theretina was significantly greater in the groups treated with RES andcompound 2 when compared to the control KI group (FIG. 12; C); theseresults show that treatment with SIRT1 activators prevented theproduction of retinal atrophy in KI mice after exposure to an intenselight source.

Evaluation of the Function of the RPE:

To evaluate the functional integrity of the RPE, the amplitude of thec-wave in the ERG was measured. The c-wave is defined as a positive slowwave that appears after the b-wave and is used to measure the functionalintegrity of the RPE. Since the amplitude of this wave can be affectedby the integrity of the photoreceptors, it is best to normalize theresults with the amplitude of the b-wave, for which reason aquantification was performed of the proportion of the amplitude of thec-wave between the amplitude of the b-wave (c-/b-wave ratio) (FIG. 13).The c-/b-wave ratio was significantly higher in the group treated withcompound 2, which suggests that the functional integrity of the RPE wasmaintained in this group.

Visual Acuity:

Finally, the optomotor test was performed in order to assess visualacuity; this test consists in evaluating the optokinetic visualfollow-up reflex that is stimulated by the rotating movement ofdifferent black and white serial horizontal bars. The response to saidvisual stimulus is manifested by a cervical tracking movement in thesame direction of the rotation as the bars. Cervical movement isrecorded with a camera, and the spatial frequency of the bars variesrandomly, as does the direction of rotation of the bars. The optomotortest was evaluated at 6 different spatial frequencies (0.031, 0.061,0.092, 0.103, 0.194, and 0.272 cycles/degree), and the spatial visionwas measured by the number of positive responses. In the group treatedwith compound 2, the number of positive responses was significantlyhigher than in control KI groups in the majority of frequencies studied(0.092, 0.103, 0.194, and 0.272 cycles/degree), and KI RES was onlysignificant at 0.194 cycles/degree (FIG. 14).

In conclusion, in the experiments described in examples 6 and 7, we havedemonstrated that the SIRT1 activator (compound 2) has a protectiveeffect on the retina of rd10 and Prpf31^(A216P/+) mice. Thisretinoprotective effect suggests that this molecule could potentially beuseful for the treatment of RP and other degenerative pathologies of theretina and RPE in which ROS, inflammation, and apoptosis occur, such asin AMD.

What is claimed is:
 1. A method of treating and/or preventing ocularpathologies, wherein the method comprises administering to a patient inneed of such treatment a therapeutically effective amount of a compoundof the general formula (I)

or any of its isomers, their pharmaceutically acceptable salts, esters,tautomers, polymorphs, or hydrates, where: R₁ is a C₁-C₂₂ alkyl group ora C₂-C₂₂ alkenyl group.
 2. The method of claim 1, wherein R₁ is a C₂-C₂₀alkyl group.
 3. The method of claim 2, wherein the compounds areselected from the group consisting of:trans-resveratrol-3-O-(6′-O-butanoyl)-β-D-glucopyranoside,trans-resveratrol-3-O-(6′-O-octanoyl)-β-D-glucopyranoside,trans-resveratrol-3-O-(6′-O-hexadecanoyl)-β-D-glucopyranoside,trans-resveratrol-3-O-(6′-O-octadecanoyl)-β-D-glucopyranoside, and anycombination thereof.
 4. The method of claim 3, wherein the compound istrans-resveratrol-3-O-(6′-O-octanoyl)-β-D-glucopyranoside.
 5. The methodof claim 1, wherein the pathologies are hereditary degenerativepathologies of the retina selected from among retinitis pigmentosa andpathologies within the group of retinal dystrophies selected from thegroup consisting of autosomal dominant retinitis pigmentosa, autosomalrecessive retinitis pigmentosa, retinitis pigmentosa linked to the Xchromosome, sporadic retinitis pigmentosa, retinitis pigmentosaassociated with other syndromes, Cokayne syndrome, cone dystrophies,cone and rod degeneration, Leber's congenital amaurosis, retinitisPunctata albescens, choroideremia, choroidal and retinal gyrate atrophy,choroidal generalized dystrophy, juvenile retinoschisis, Wagner'svitreoretinal degeneration, autosomal dominant vitreoretinalchoroidopathy, fundus albipunctatus, Stargardt's disease, Bestvitelliform macular dystrophy, Usher syndrome, Bardet-Biedl syndrome,fenestrated macular dystrophy, Sorsby pseudoinflammatory maculardystrophy, and/or dominant drusen.
 6. The method of claim 1, wherein theocular pathologies are age-related macular degeneration.
 7. The methodof claim 1, wherein the ocular pathologies are degenerative inflammatorypathologies of the retina and of the optic nerve.
 8. The method of claim1, wherein the ocular pathologies are ocular pathologies that increaseintraocular pressure and that involve degeneration of the ganglion cellsof the retina and atrophy of the optic nerve selected from the groupconsisting of open-angle and closed-angle glaucoma.
 9. The method ofclaim 1, wherein the ocular pathologies are neurological pathologiesthat are accompanied by degenerative lesions of the retinal or opticnerve selected from the group consisting of amyotrophic lateralsclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease,Graefe syndrome, Hallgren syndrome, Hallen Vorden Spatz syndrome,autosomal dominant cerebellar ataxia, and pigmentary degeneration of theretina.
 10. The method of claim 1, wherein the compound of formula (I)is administered together with a controlled-release system.
 11. Themethod of claim 10, wherein the controlled-release system is acyclodextrin.
 12. The method of claim 11, wherein the cyclodextrin is2-hydroxypropyl β-cyclodextrin.
 13. A pharmaceutical compositioncomprising at least one compound of the general formula (I):

or any of its isomers, their pharmaceutically acceptable salts, esters,tautomers, polymorphs, or hydrates, where: R₁ is a C₁-C₂₂ alkyl group ora C₂-C₂₂ alkenyl group; and a cyclodextrin.
 14. The composition of claim13, wherein the compound of formula (I) is selected from amongtrans-resveratrol-3-O-(6′-O-butanoyl)-β-D-glucopyranoside,trans-resveratrol-3-O-(6′-O-octanoyl)-β-D-glucopyranoside,trans-resveratrol-3-O-(6′-O-hexadecanoyl)-β-D-glucopyranoside,trans-resveratrol-3-O-(6′-O-octadecanoyl)-β-D-glucopyranoside, and anycombination thereof.
 15. The composition of claim 13, wherein thecyclodextrin is 2-hydroxypropyl β-cyclodextrin.